MRNA (messenger ribonucleic acid) production device

Abstract

The invention belongs to the technical field of mRNA production and manufacturing, and discloses an mRNA production device. The mRNA production device provided by the invention comprises a production tank filled with heat conduction liquid, a linkage assembly arranged in the production tank, a flexible reaction bag and a solution disturbance assembly, the linkage assembly comprises an elastic connecting piece and a storage plate, and the elastic connecting piece is connected with the peripheral wall of the production tank and enables the storage plate to be suspended in the production tank; the flexible reaction bag is arranged on the object placing plate, the solution disturbance assembly drives the heat conduction liquid to flow so as to drive the object placing plate to shake, and the solution disturbance assembly keeps a non-contact state with the flexible reaction bag and the object placing plate. According to the mRNA production device provided by the invention, the contradiction between the uniformity of a reaction system and the structural integrity of mRNA in the existing IVT reaction amplification is effectively solved, and the damage to the mRNA structure can be reduced while the good uniformity of the IVT reaction system is kept, so that a large amount of high-quality mRNA is prepared, and the industrial amplification production of mRNA is favorably realized.

Description

Technical Field

[0001] The invention belongs to the technical field of mRNA production and manufacturing, and in particular relates to an mRNA production device. Background Art

[0002] Messenger RNA (mRNA) is a single-stranded RNA molecule that can efficiently translate the genetic information it carries into corresponding proteins on the ribosomes in cells. mRNA vaccines and mRNA drugs carry RNA molecules encoding specific antigenic proteins through a specific delivery system, express the corresponding antigens in the body or adjust the gene expression in the cells, thereby stimulating the body to produce specific immune responses or change the body's metabolism to achieve the effect of disease prevention and / or treatment. mRNA vaccines and mRNA drugs respond quickly to changes in diseases and pathogens and are highly safe, so they have broad application prospects.

[0003] At present, the production of mRNA mainly includes the design and production of linear plasmids, in vitro transcription (IVT) of mRNA, purification of mRNA and packaging. Among them, IVT is a key step in mRNA production, which involves transcribing and synthesizing mRNA using linear plasmids as templates in a cell-free system. In the amplification of the IVT reaction, in order to ensure the uniformity of the reaction system to achieve better preparation effects, the reaction system needs to be mixed. However, in the amplified IVT reaction system, traditional mixing techniques often cause the solution to produce large internal shear forces, causing serious damage to the structure of mRNA, resulting in the failure of preparation, limiting the large-scale industrial production of mRNA, and having great limitations. Summary of the invention

[0004] The purpose of the present invention is to solve the contradiction between the uniformity of the reaction system and the integrity of the mRNA structure in the existing IVT reaction amplification, and to provide an mRNA production device. The mRNA production device can reduce the damage to the mRNA structure while maintaining the good uniformity of the IVT reaction system, thereby preparing a large amount of high-quality mRNA, which is conducive to the industrial amplification of mRNA production.

[0005] Specifically, the mRNA production device provided by the present invention includes: a production tank, in which a heat-conducting liquid is arranged; a linkage component, which is arranged in the production tank, and the linkage component includes an elastic connector and a storage plate, the elastic connector is connected to the peripheral wall of the production tank, and the storage plate is connected to the elastic connector to be suspended in the production tank; a flexible reaction bag, the flexible reaction bag is arranged on the storage plate, and the flexible reaction bag and the storage plate are immersed in the heat-conducting liquid; a solution disturbance component, the solution disturbance component is used to drive the heat-conducting liquid to flow and drive the storage plate to shake, and the solution disturbance component maintains a non-contact state with the flexible reaction bag and the storage plate.

[0006] Furthermore, the production tank includes a tank body and a top cover; the top cover is connected to one end of the elastic connector, and the storage plate is connected to the other end of the elastic connector, so that the elastic connector is in a stretched state.

[0007] Furthermore, the bottom wall of the production tank is connected to one end of the elastic member, and the storage plate is connected to the other end of the elastic connecting member, so that the elastic connecting member is in a compressed state.

[0008] Furthermore, the storage plate is arranged at an angle.

[0009] Furthermore, the storage plate is a grid plate.

[0010] Furthermore, the solution disturbance component is a magnetic stirring component, which includes a magnetic stirring table and a magnetic rotor. The production tank is arranged on the magnetic stirring table. The magnetic rotor is arranged in the production tank to drive the flow of the thermal transfer liquid, and the magnetic rotor maintains a non-contact state with the flexible reaction bag and the storage plate.

[0011] Furthermore, the flexible reaction bag is arranged on a side of the storage plate away from the magnetic rotor.

[0012] Furthermore, the solution disturbance component is a mechanical stirring component, which includes a stirring rod and a stirring motor. The stirring motor drives the stirring rod to rotate. The stirring rod is inserted into the production tank to drive the flow of the thermal transfer liquid, and the stirring rod maintains a non-contact state with the flexible reaction bag and the storage plate.

[0013] Furthermore, the flexible reaction bag is arranged on a side of the storage plate away from the stirring rod.

[0014] Furthermore, a temperature controller is provided in the production tank.

[0015] Beneficial effects:

[0016] The mRNA production device provided by the present invention comprises a production tank containing a heat-conducting liquid, a linkage component arranged in the production tank, and a solution disturbance component for driving the flow of the heat-conducting liquid, wherein the linkage component comprises an elastic connector and a placement plate suspended in the production tank by the elastic connector, and a flexible bag for containing an IVT reaction liquid is arranged on the placement plate. During the mRNA preparation process, the solution disturbance component works to make the heat-conducting liquid in the production tank flow, and the kinetic energy generated by the flow of the heat-conducting liquid indirectly acts on the flexible reaction bag through the placement plate to make it shake, and the elastic connector connected to the placement plate will generate a reaction force applied to the placement plate when subjected to the dual force of the heat-conducting liquid and the placement plate, changing the shaking state of the placement plate to optimize the force generated by the placement plate on the flexible reaction bag, so that the shaking of the flexible reaction bag can achieve full mixing of the reaction liquid while maintaining the integrity of the mRNA, so as to effectively solve the contradiction between the uniformity of the reaction system and the structural integrity of the mRNA in the existing IVT reaction amplification, which is conducive to the large-scale industrial production of mRNA and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the structure of the mRNA production device provided in the first embodiment of the present invention;

[0018] Figure 2 for Figure 1 Sectional view of AA in the middle;

[0019] Figure 3 A schematic diagram of the structure of an mRNA production device provided in the second embodiment of the present invention;

[0020] Figure 4 for Figure 3 Cross-sectional view of BB.

[0021] Figure numerals: 1. production tank; 11. tank body; 12. top cover; 2. first linkage assembly; 21. spring; 22. first storage plate; 3. flexible reaction bag; 4. magnetic stirring assembly; 41. magnetic stirring table; 42. magnetic rotor; 5. temperature controller; 6. second linkage assembly; 61. U-shaped spring sheet group; 62. second storage plate; 7. mechanical stirring assembly; 71. stirring rod; 72. stirring motor. DETAILED DESCRIPTION

[0022] Although the present invention can be easily embodied in different forms of embodiments, only some of the specific embodiments are shown in the drawings and will be described in detail in this specification. It should be understood that this description should be regarded as an exemplary illustration of the principles of the invention and is not intended to limit the invention to that described herein.

[0023] Thus, a feature indicated in this specification will be used to illustrate one of the features of an embodiment of the present invention, rather than implying that each embodiment of the present invention must have the described feature. In addition, it should be noted that this specification describes many features. Although some features can be combined together to illustrate possible system designs, these features can also be used in other combinations that are not explicitly described. Thus, unless otherwise stated, the described combinations are not intended to be limiting.

[0024] In the embodiments shown in the drawings, the indications of directions (such as up, down, left, right, front and back) used to explain the structure and movement of various elements of the present invention are not absolute but relative. These descriptions are appropriate when these elements are in the positions shown in the drawings. If the description of the positions of these elements changes, the indications of these directions also change accordingly.

[0025] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0026] See also Figure 1 and Figure 2 . Figure 1 It is a schematic diagram of the structure of the mRNA production device of the first embodiment of the present invention. Figure 2 yes Figure 1 The mRNA production device provided in the first embodiment includes: a production tank 1, a first linkage component 2, a flexible reaction bag 3 and a magnetic stirring component 4.

[0027] The production tank 1 includes a tank body 11 and a top cover 12, and the tank body 11 contains a heat transfer liquid. The first linkage assembly 2 includes a spring 21 and a first storage plate 22, one end of the spring 21 is fixedly connected to the inner wall of the top cover 12, and the other end is fixedly connected to a clip, and the clip is used to clamp and fix the first storage plate 22, so that the first storage plate 22 is immersed in the heat transfer liquid in the tank body 11 and remains suspended, and the spring 21 is in a stretched state at this time. The flexible reaction bag 3 is relatively fixed to the upper surface of the first storage plate 22 by a fixing rope, so that the flexible reaction bag 3 can shake with the shaking of the first storage plate 22.

[0028] The magnetic stirring assembly 4 includes a magnetic stirring table 41 and a magnetic rotor 42. The production tank 1 is placed on the magnetic stirring table 41, and the magnetic rotor 42 is placed at the bottom of the production tank 1. The magnetic rotor 42 stirs the thermal fluid to make it flow under the drive of the magnetic stirring table 41, and the magnetic rotor 42 maintains a non-contact state with the flexible reaction bag 3 and the first storage plate 22 during the rotation process.

[0029] A temperature controller 5 is also installed in the tank body 11, and the temperature controller 5 is used to detect and control the temperature of the heat transfer liquid in the tank body 11, so that during the IVT reaction, the reaction temperature is in a relatively constant state, so as to achieve a better mRNA preparation effect.

[0030] In the present invention, the number of springs 21 in the first linkage assembly 2 can be adaptively selected according to the placement state of the first storage plate 22, and the specific number can be 1, 2, 3, 4 or any integer greater than them, and those skilled in the art can adaptively select the number of springs 21 and the connection method and fixed position of the springs 21 and the first storage plate 22 according to the needs, and the present invention does not impose any special restrictions on them. That is, Figure 2 The number of springs 21, the connection method with the first placement plate 22 and the connection position shown in the figure are only one example (first embodiment) of the mRNA production device of the present invention, but should not be understood as limiting the scope of protection of the present invention.

[0031] In the present invention, the first placement plate 22 is limited to placing and fixing the flexible reaction bag 3, and the present invention does not impose any special restrictions on its specific shape and structure. In the first embodiment, the first placement plate 22 is preferably a mesh plate, so as to effectively increase the contact area between the flexible reaction bag 3 and the thermal conductive liquid while being able to effectively fix the flexible reaction bag 3.

[0032] In the present invention, the first placement plate 22 is kept suspended in the tank body 11 to give it the ability to shake with the flow of the thermal fluid in the tank body 11. Specifically, it can be placed horizontally in the tank body 11, vertically in the tank body 11, or tilted in the tank body 11. In this first embodiment, the first placement plate 22 is preferably placed tilted in the tank body 11 so that the flexible reaction bag 3 is also in a tilted state, which can further optimize the force on the flexible reaction bag 3, while achieving a good mixing effect on the reaction liquid in the flexible reaction bag 3, reducing the damage to the mRNA.

[0033] In the present invention, the relative fixing method of the flexible reaction bag 3 and the first storage plate 22 shown in the first embodiment is only an example. Those skilled in the art can select a suitable fixing method from the prior art to achieve the fixation between the flexible reaction bag 3 and the first storage plate 22 according to actual needs, and the present invention does not impose any special limitation on it.

[0034] In the present invention, the heat transfer liquid contained in the tank body 11 is a type of liquid with heat transfer function commonly used in the prior art, and it is non-corrosive to the various components in the mRNA production device. Those skilled in the art can make adaptive choices according to the actual needs of use. The present invention does not impose any special restrictions on it. Specific examples can be but are not limited to: water and / or heat transfer oil. Among them, the heat transfer liquid contained in the tank body 11 provided in the first embodiment is water.

[0035] In the present invention, the temperature controller 5 is used to monitor and adjust the temperature of the heat transfer liquid in the tank body 11. It is a type of temperature detection and control device commonly used in the prior art. Those skilled in the art can make adaptive choices according to actual needs, and the present invention does not impose any special limitations on it.

[0036] In the first embodiment, when the mRNA production device is used to perform an amplified IVT reaction of mRNA, the raw materials required for the reaction are added to the flexible reaction bag 3 and fixed on the first placement plate 22, the first placement plate 22 is suspended in the tank body 11 by the clip on the spring 21, and an appropriate amount of water is added to the tank body 11 to completely submerge the first placement plate 22 and the flexible reaction bag 3; then the magnetic stirring table 41 is turned on to rotate the magnetic rotor 42 in the tank body 11 at a speed of 5 to 200 rpm to stir the water flow, and the internal shear force generated by the water flow acts on the first placement plate 22 to cause it to shake, and the elastic force of the spring 21 in the stretched state can play a buffering role, optimize the shaking state of the first placement plate 22, and use the transmission effect of the first placement plate 22 to make the internal shear force generated by the water flow indirectly act on the flexible reaction bag 3 to mix the reaction solution therein, and the damage to the mRNA is small. At the same time, the rotation of the magnetic rotor 42 is also conducive to maintaining the uniformity of the solution temperature at various locations in the tank body 11.

[0037] See also Figure 3 and Figure 4 . Figure 3 It is a schematic diagram of the structure of the mRNA production device of the second embodiment of the present invention. Figure 4 yes Figure 3 The mRNA production device provided in the second embodiment includes: a production tank 1, a second linkage component 6, a flexible reaction bag 3 and a mechanical stirring component 7.

[0038] The production tank 1 includes a tank body 11 and a top cover 12. A temperature controller 5 is installed in the tank body 11 and contains a heat transfer liquid. The second linkage assembly 6 includes a U-shaped spring sheet group 61 and a second storage plate 62. The U-shaped spring sheet group 61 is formed by a plurality of U-shaped springs connected in series, and one side is placed at the bottom of the tank body 11, and the other side is fixedly connected to the second storage plate 62, so that the second storage plate 62 is immersed in the heat transfer liquid in the tank body 11 and remains suspended, and the U-shaped spring sheet group 61 is in a compressed state at this time. The flexible reaction bag 3 is relatively fixed to the lower surface of the second storage plate 62 by a fixing rope, so that the flexible reaction bag 3 can shake with the shaking of the second storage plate 62.

[0039] The mechanical stirring assembly 7 includes a stirring rod 71 and a stirring motor 72 . The stirring rod 71 passes through the top cover 12 , and the stirring head of the stirring rod 71 is immersed in the thermal liquid and maintains a non-contact state with the flexible reaction bag 3 and the second storage plate 62 .

[0040] In the second embodiment, when the mRNA production device is used to perform an amplified IVT reaction of mRNA, the raw materials required for the reaction are added to the flexible reaction bag 3 and fixed on the second placement plate 62, the second placement plate 62 is suspended in the tank body 11 through the U-shaped spring sheet group 61, and an appropriate amount of water is added to the tank body 11 to completely submerge the second placement plate 62 and the flexible reaction bag 3; then the stirring motor 72 is turned on to rotate the stirring rod 71 at a speed of 5 to 200 rpm to stir the water flow, and the internal shear force generated by the water flow acts on the second placement plate 62 to cause it to shake, and the elastic force of the U-shaped spring sheet group 61 in the compressed state can play a buffering role, optimize the shaking state of the second placement plate 62, and use the transmission effect of the second placement plate 62 to make the internal shear force generated by the water flow indirectly act on the flexible reaction bag 3 to mix the reaction solution therein, and the damage to the mRNA is small. At the same time, the rotation of the stirring rod 71 is also conducive to maintaining the uniformity of the solution temperature at various locations in the tank body 11.

[0041] The mRNA production apparatus provided in the first embodiment and the second embodiment was used to prepare mRNA. The total volume of the reaction solution was 20 mL, and the reaction raw materials, addition amounts, and reaction conditions were specifically shown in Table 1.

[0042] Table 1.

[0043]

[0044] The DNA template includes a nucleotide fragment whose sequence is shown in SEQ ID NO: 1, as shown in Table 2.

[0045] Table 2.

[0046]

[0047]

[0048] Take 50 μL of the final IVT reaction solution, use an RNA purification kit (NEB, catalog number T2050L) to obtain the purified mRNA product according to the instructions, and use the RNA mode of the Nanodrop microspectrophotometer to detect the total amount of mRNA in the IVT reaction solution (in μg), and calculate the mRNA yield (mg / mL) of 50 μL of the IVT reaction solution provided in each embodiment according to the following formula.

[0049] mRNA yield (mg / mL) = X*21 / 1000

[0050] Wherein, X is the total amount of mRNA obtained after 50 μL of IVT reaction solution was sampled and purified. The results are shown in Table 3.

[0051] Table 3.

[0052] Group mRNA yield (mg / mL) Example 1 4.9 Example 2 5.1

[0053] From the test results shown in Table 3, it can be seen that the mRNA production device provided by Examples 1 and 2 of the present invention can effectively solve the contradiction between the uniformity of the reaction system and the structural integrity of mRNA in the existing IVT reaction amplification, and the mRNA yields are 4.9 mg / mL and 5.1 mg / mL, respectively, achieving the preparation of milligram-level mRNA, which is conducive to the industrial amplification production of mRNA and has good application prospects.

[0054] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and purpose of the present invention.

Claims

1. An mRNA production device, characterized in that: The mRNA production device comprises: A production tank, wherein a heat transfer liquid is arranged in the production tank; A linkage assembly, the linkage assembly is arranged in the production tank, the linkage assembly comprises an elastic connector and a storage plate, the elastic connector is connected to the peripheral wall of the production tank, and the storage plate is connected to the elastic connector to be suspended in the production tank; A flexible reaction bag, wherein the flexible reaction bag is arranged on a storage plate, and the flexible reaction bag and the storage plate are immersed in a heat transfer liquid; A solution disturbance component is used to drive the thermal fluid to flow and drive the placement plate to shake, and the solution disturbance component maintains a non-contact state with the flexible reaction bag and the placement plate.

2. The mRNA production device according to claim 1, characterized in that The production tank comprises a tank body and a top cover; the top cover is connected to one end of an elastic connecting member, and the storage plate is connected to the other end of the elastic connecting member, so that the elastic connecting member is in a stretched state.

3. The mRNA production device according to claim 1, characterized in that The bottom wall of the production tank is connected to one end of the elastic member, and the storage plate is connected to the other end of the elastic connecting member, so that the elastic connecting member is in a compressed state.

4. The mRNA production device according to claim 1, characterized in that The storage plate is arranged tilted.

5. The mRNA production device according to claim 1, characterized in that The storage plate is a grid plate.

6. The mRNA production device according to claim 1, characterized in that The solution disturbance component is a magnetic stirring component, which includes a magnetic stirring platform and a magnetic rotor. The production tank is arranged on the magnetic stirring platform. The magnetic rotor is arranged in the production tank to drive the flow of the thermal transfer liquid, and the magnetic rotor maintains a non-contact state with the flexible reaction bag and the storage plate.

7. The mRNA production device according to claim 6, characterized in that The flexible reaction bag is arranged on a side of the storage plate away from the magnetic rotor.

8. The mRNA production device according to claim 1, characterized in that The solution disturbance component is a mechanical stirring component, which includes a stirring rod and a stirring motor. The stirring motor drives the stirring rod to rotate. The stirring rod is inserted into the production tank to drive the flow of the thermal transfer liquid, and the stirring rod maintains a non-contact state with the flexible reaction bag and the storage plate.

9. The mRNA production device according to claim 8, characterized in that The flexible reaction bag is arranged on a side of the storage plate away from the stirring rod.

10. The mRNA production device according to claim 1, characterized in that: A temperature controller is arranged in the production tank.

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